The recent developments in plant genetic engineering allow routine introduction of recombinant DNA in a wide range of plants. Transcription and translation was observed for most of the chimeric genes, however suboptimal expression is often encountered when expression of AT-rich genes is attempted. One of the prime examples of such difficulties was the expression of Bt ICPs.
Numerous publications teach the expression of different Bt ICPs in a wide range of plant species. Truncating the Bt ICP genes so as to encode a smaller and more soluble protein that retained full toxicity was found to be critical to obtain insect controlling amounts of Bt ICP in the plants [Vaeck et al., Nature, 328: 33-37 (1987); Fischhof et al., Bio/Technology 5: 807-813 (1987); Carozzi et al., Plant Molecular Biology 20: 539-548 (1992)].
Subsequent publications described the enhancement of the expression levels of Bt ICP genes in plant species, in order to be able to target also less susceptible insect species. Different approaches were followed to modify the introduced bacterial DNA sequences encoding Bt ICPs to avoid the presence of sequences that could negatively affect expression in the plant cells. To this end, nucleic acid sequences were provided that encode a Bt ICP with essentially the same amino acid sequence as an existing Bt ICP but wherein one or more of the following modifications were included:
the nucleic acid sequence surrounding the translation initiation codon was changed to resemble more the translation initiation sequences preferably used by plants. PA1 the overall codon usage was modified to better reflect the preferred codon usage of a particular plant species. PA1 cryptic promoter signals were removed. PA1 nucleic acid sequences that target the hnRNA into an abortive splicing pathway were eliminated. PA1 potential termination signals for DNA-dependent RNA polymerase II within the coding sequence were removed. PA1 putative mRNA destabilizing sequences were replaced. PA1 presumptive alternative polyadenylation sites were avoided. PA1 a.) a first promoter recognized by a DNA-dependent RNA polymerase different from a eukaryotic RNA polymerase II, particularly a T3 or T7 RNA polymerase specific promoter; PA1 b.) a DNA region encoding a chimeric RNA which comprises a 5' UTR, a heterologous coding sequence, preferably an AU-rich coding sequence, and a 3' UTR; and optionally PA1 c.) a terminator sequence recognized by said RNA polymerase PA1 i) a first translation enhancing sequence derived from the 5' region of genomic or subgenomic RNA of a positive stranded RNA plant virus, preferably a necrovirus, especially STNV-2 or TNV-A, located in the 5' region of the chimeric RNA; PA1 ii) a second translation enhancing sequence derived from the 3' region of genomic or subgenomic RNA of a positive-stranded RNA plant virus, preferably a necrovirus, especially STNV-2 or TNV-A, located in the 3' region of the chimeric RNA; PA1 a.) a second plant-expressible promoter; PA1 b.) a DNA sequence encoding a single subunit bacteriophage RNA polymerase such as a T3 or T7 RNA polymerase functionally linked to a nuclear localization signal; PA1 a.) transforming the nuclear genome of a plant cell with the above-mentioned chimeric genes; and PA1 b.) regenerating a transformed plant from the transformed cell.
[Perlak et al., Proc. Natl. Acad Sci. USA 88: 3324-3328 (1991); Adang et al., Plant Mol. Biol. 21: 1131-1145 (1993), Murray et al. Plant Mol. Biol. 116: 1035-1050 (1991) WO 91/16432, WO 93/09218].
Recently, Mc Bride et al. described the introduction of a native Bt ICP coding sequence under control of a T7 promoter or a plastid expression signal in the chloroplasts of tobacco plants in an attempt to circumvent the problem of poor expression of full-length protoxin genes from the nucleus of plants, particularly those with a high AT-content. The regenerated plants from these transplastomic lines were reported to express Bt ICP at a high level in mature leaves using the prokaryotic-like transcriptional and translational machinery of the plastid (Mc Bride et al., Bio/Technology 13: 362-365 (1995); WO 95/24492, WO 95/24493). However, the transformation process set forth in these references is complicated because it requires the use of plastid transformation vectors and/or the transport of appropriate polymerases from the cytoplasm to the chloroplasts. Furthermore, the references remain silent on the level of ICPs in tissues other than mature leaves, such as root or stem tissue which constitute important targets for pests such as corn root worm (Diabrotica spp), European corn borer (Ostrinia nubilalis) or cutworms (e.g., Agrotis spp.).
Unique features of eukaryotic mRNA are the presence of the m.sup.7 G cap at its 5' end and a 3' poly(A) tract. Several functions at different stages of gene expression have been attributed to the cap at the 5' end, which is added shortly after transcription elongation has started, including a role in RNA stabilization, splicing, transport and translation. The cap structure supposedly binds to the translation initiation factor elF-4F, allowing the ribosomal subunits and proper factors to bind and initiate at the first AUG codon in a favourable sequence context. Absence of this 5' cap structure in naturally capped plant viral RNA or cellular mRNA decreases the translational efficiency substantially [Fletcher et al, J. Biol. Chem. 265: 19582-19587 (1990)].
A role for the poly(A) tail found at the 3' end of most eukaryotic mRNAs has been implied in mRNA stability, its transport into the cytoplasm, and its efficient translation [Jackson and Standart, Cell, 62: 15-24,1990]. The poly(A) tail, complexed with poly(A)-binding protein is believed to enhance the formation of 40S translational initiation complexes, presumably through promoting some sort of interaction between 5' and 3'-proximal elements of the mRNA [Tarum and Sachs, Genes and Dev. 9: 2997-3007 (1995)].
Whereas the majority of eukaryotic mRNAs have capped 5' ends and poly(A) tails at the 3' ends, the genomic or subgenomic RNAs of plant viruses often lack one or both. For positive-strand RNA viruses, the RNAs are translated early upon infection, even though cellular templates are prevalent. It is often due to the presence of alternative terminal structures that viral RNA templates exhibit high translational efficiency.
U.S. Pat. No. 4,820,639 describes a process and means for increasing production of protein translated from eukaryotic messenger ribonucleic acid comprising transferring a regulatory nucleotide (nt) sequence from a viral coat protein mRNA to the 5' terminus of a gene or complementary deoxyribonucleic acid (cDNA) encoding the protein to be produced to form a chimeric DNA sequence.
U.S. Pat. No. 5,489,527 and the European patent publication (EP) 0270611 both describe the use of 5' regions of RNA viruses as enhancers of translation of mRNA, especially 5' regions derived from plant RNA viruses.
Publication of the PCT patent application (WO) 91/00905 and U.S. Pat. No. 5,135,855 describe the use of untranslated regions from an encephalomyocarditis virus to confer cap-independent translation to RNAs in mammalian cells, particularly when a prokaryotic transcription system is used in these eukaryotic cells.
EP 0589841 provides a dual method for producing male-sterile plants, as well as compositions and methods for high level expression of a coding region of interest in a plant by expression of a T7 RNA polymerase in a plant cell that contains a second expression cassette comprising a T7 5' regulatory region linked to the coding region of interest.